Thermostable variants of human interferon-γ(IFN-γ)

ABSTRACT

The invention provides new variants of recombinant human interferon-γ (rhIFN-γ), vectors and host cells for their production, and therapeutic methods employing them. The variants are characterized by the substitution of one or more pairs of amino acids selected from Glu 8  -Ser 70 , Ala 18  -His 112 , Lys 81  Leu 121 , and Gln 49  -Leu 96  by pairs of Cys residues, and optionally by the deletion of from one to ten amino acid residues from C-terminus of the native IFN-γ sequence. The variants of the invention exhibit greater thermal stability and no loss of biological activity as compared to native-sequence rhIFN-γ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national application of internationalapplication Ser. No. PCT/DE96/01556, filed Aug. 15, 1996, which claimspriority to German serial No.195 35 853.8, filed Sep. 18, 1995.

The invention relates to variants of the human recombinant interferon-γwith increased thermal stability.

Interferon-γ is suitable as an antiviral, antiproliferative,immunomodulatory human therapeutic agent, particularly for the treatmentof kidney tumours and chronic granulomatosis.

Like the interleukins, interferons belong to the class of the cytokinesand are listed in various classes: interferon-α, interferon-β,interferon-γ, interferon-ω and interferon-τ. Interferon-γ is aglycoprotein, whose amino acid sequence has been known since 1982 (Nucl.Acid Res. 10, pages 2487 and following (1982)). In the mature conditionthe interferon-γ has 143 amino acids and a molecular weight of 63 to 73kilodaltons. The amino acid sequence of interferon-γ is shown in FIG. 1.It will be noticed that the sequence contains no cystein.

The tertiary and quaternary structure of the non-glycosylised proteinwas clarified in 1991 (Science 252, page 698 and following (1991)).According to this, interferon-γ exists as a homodimer, the monomersbeing orientated in contrary directions in such a way that theC-terminal end of one monomer is located in the vicinity of theN-terminal end of the other monomer. Each of these monomers in all hassix α-helices.

Interferon-γ is also called immunointerferon, as it has non-specificantiviral, antiproliferative and in particular immunomodulatory effects.Its production in T-helper-lymphocytes is stimulated by mitogens andantigens. The effect of the expressed interferon-γ has not yet beenprecisely clarified, but is subject to intensive research. Inparticular, interferon-γ leads to the activation of macrophages and tothe synthesis of histocompatability antigens of the class 2. In vitro,the activity of interferon-γ is normally determined as a reduction inthe virus-induced cytopathic effect, which arises from treatment withinterferon-γ. Due to its antigen-non-specific antiviral,antiproliferative and immunomodulatory activity it is suitable as ahuman therapeutic agent, for example of kidney tumours and chronicgranulomatosis. Clinical studies of various tumours are being carriedout at present. Great expectations are set also on the interferon-γtherapy of neurodermitis. Furthermore, interferon-γ is used in theresearch field also as a fine chemical for example for the stimulationof cell cultures or for determining interferon levels.

Since 1982 it has been possible to express variants of the humaninterferon-γ in bacteria such as escherichia coli (Nature 295, pages 503and following (1982)). The monomer of these recombinant variants of thehuman interferon-γ has, in addition to the 143 amino acids of the nativehuman monomer, an additional N-terminal methionin as an additional aminoacid. This recombinant human interferon-γ (rhu-IFN-γ) is notglycosylised. It can be obtained in large quantities and was thereforeextremely accurately characterised.

The recombinant human interferon-γ also exists as a dimer and has normalbiological activity. It is acid-sensitive and temperature-sensitive witha melting point of 52° C.

Of all the interferons, the thermal stability of human interferon-γ isthe lowest. This low thermal stability of interferon-γ renders itdifficult to use as a human therapeutic agent.

Tests were carried out at an early stage in order to generate variantswith improved properties of the recombinant human interferon-γ with theaid of molecular-biological methods. In EP 0 306870 A2, variants ofrecombinant human interferon-γ were presented, whose activity wassignificantly increased by splitting off the C-terminal 7-11 aminoacids. In addition, WO 92-08737 indicates a variant of recombinant humaninterferon-γ (Interferon-γ C-10L) which has an increased biologicalactivity. However, in none of these variants of recombinant interferon-γwas the thermal stability significantly increased. Therefore the lowthermal stability also renders difficult the use of these proteins as ahuman therapeutic agent.

The object of the present invention is to make available variants of therecombinant human interferon-γ whose thermal stability is increased, thebiological activity being similar, preferably equal to or even betterthan that of the unaltered recombinant human interferon-γ. It is afurther object of this invention to indicate gene sequences, vectorswith gene sequences and micro-organisms with gene sequences which can beused to manufacture the expression system and the variant according tothe invention of interferon-γ.

This object is achieved in that there are indicated DNA sequences andrecombinant vectors with such DNA sequences, for example plasmids andbacteriophages, and micro-organisms which contain such DNA sequences,which code for polypeptides, which are characterised in comparison torecombinant human interferon-γ by increased thermal stability.Furthermore, methods are indicated by means of which such DNA sequences,recombinant vectors and micro-organisms can be produced. With the aid ofthe oligonucleotides according to the invention and correspondingrestriction endonucleases, on the basis of a plasmid known per se, whichcontains the gene for recombinant human interferon-γ, recombinantvectors such as plasmids or bacteriophages and micro-organisms can beproduced, which contain a DNA sequence which codes for the polypeptidesaccording to the invention.

There is further indicated a manufacturing method for the proteinsaccording to the invention, by means of which, with the aid of themicro-organisms according to the invention the proteins according to theinvention can be obtained, concentrated and cleaned up, and uses of theproteins according to the invention as a human therapeutic agent or/andfine chemical.

The polypeptides according to the invention consist of a sequence of 144amino acids, made up of the sequence of the 143 amino acids of therecombinant human interferon-γ and an additional N-terminal methionin.Otherwise the polypeptide according to the invention differs from themonomer of the recombinant human interferon-γ in that at least one pairof amino acids from four predetermined amino acid pairs is exchanged forcystein. These four amino acid pairs are Glu8 and Ser70, Ala18 andHis112, Lys81 and Leu121 and Gln49 and Leu96. The complete amino acidsequence is given for the recombinant human interferon-γ in FIG. 1 andfor the polypeptide according to the invention in FIG. 2. In this caseXaa means Glu8, Xab Ser70, Xba Ala18, Xbb His112, Xca Lys81, Xcb Leu121,Xda Gln49 and Xdb Leu 96, insofar as these amino acids do not stand forcystein in the polypeptide according to the invention.

It became surprisingly apparent that the proteins according to theinvention have higher thermal stability in comparison to recombinanthuman interferon-γ.

The proteins according to the invention are probably, like recombinanthuman interferon-γ, homodimers, whose monomers are so oriented that theN-terminal end of one monomer is located in the vicinity of theC-terminal end of the other monomer. Possibly the exchange in pairs ofthe amino acids leads to additional intermonomeric and intramonomericdisulphide bridges. Thus the exchange of Ala18 and His112 and theexchange of Lys80 and Leu120 probably leads to intermonomeric disulphidebridges, and the exchange of Glu8 and Ser70 to an intramonomericdisulphide bridge, while the exchange of Glu49 and Leu96 does not leadto any additional covalent bond. Clearly the introduction of cysteinpairs into the amino acid sequence of the polypeptide monomer cannotlead to a direct conclusion regarding the formation of disulphidebridges.

Due to the improved properties of the proteins and polypeptidesaccording to claims 1, 2 and 3 compared to the previous recombinanthuman interferon-γ, these proteins are particularly suitable for variousforms of application as a human therapeutic agent. They are usable withadvantage particularly in those areas in which previously recombinanthuman interferon-γ was used as a medicinal drug, such for example askidney tumours or chronic granulomatosis. It is further to be expectedthat the proteins according to the invention can be used in all futureindications in which previous recombinant human interferon-γ will beused as a medicinal drug.

The variants according to the invention of the recombinant humaninterferon-γ are also suitable for use as fine chemical for example forin vitro tests, in order to determine interferon levels and for thestimulation of cell cultures. Due to their higher thermal stability andthus their improved handling, and due to their antiviral,antiproliferative and immunomodulatory biological activity, they canreplace previous recombinant human interferon-γ in all areas where thelatter is used.

Further advantageous embodiments are illustrated in the dependentclaims.

A shortening of the C-terminal end of the polypeptide monomer by one toten amino acids leads to an improvement in the biological activity. Inthis way both the thermal stability of the variants according to theinvention of the recombinant human interferon-γ and also its biologicalactivity are improved in comparison to recombinant human interferon-γ.

The production of the new recombinant vectors and micro-organisms willbe described in the following.

In order to produce a recombinant micro-organism which contains a genesequence which codes for one of the polypeptides according to theinvention, the sequence of the human recombinant interferon-γ is cutwith the aid of restriction endonucleases from a vector which containsthis sequence, and cloned into the DNA of a phage. The phage DNA is thenmutated with the aid of appropriate oligonucleotides, so that it codesfor one of the polypeptides according to the invention. The geneticallyaltered phage is then multiplied and the altered sequence for humanrecombinant interferon-γ is cut out of the DNA of the phage with the aidof the same restriction endonucleases, and cloned back into the plasmid.With the aid of this plasmid, a micro-organism is now transfected, whichcan then express the new gene coding for the polypeptide according tothe invention.

This method will now be illustrated by the example of a transfectedbacterium escherichia coli K12 strain JM 105.

The known plasmid pKK-233-2/IFN-γ was used as an outset material. Thisplasmid was digested with the restriction endonucleases EcoRI andHindIII. This plasmid has between the two intersections for EcoRI andHindIII the gene for human recombinant interferon-γ and a strongIPTG-inducable trc-promoter. This DNA section with a length of 751 basepairs was then likewise cloned with EcoRI and HindIII into the likewiseknown bacteriophage M13mp18. Then the DNA of the bacteriophage wasmutated with methods known per se and appropriate oligonucleotides insuch a way that the gene for interferon-γ codes for one of the variantsaccording to the invention. This new bacteriophage M13mp18/IFN-γ wasmultiplied in the bacterium escherichia coli K12 strain TG1. The nowaltered sequence section, which contained the gene for the alteredrhu-IFN-γ was cut out from the c-DNA of the multiplied phage with theaid of the same restriction endonucleases EcoRI and HindIII, and clonedback into the plasmid pKK233-2. This now likewise altered plasmidpKK233-2/IFN-γ was now used to transfect the bacterium escherichia coliK12 strain JM105.

The oligonucleotides used have a length of 29 base pairs and a sequencewhich is complementary to the single-strand DNA sequence of thecorresponding bacteriophage DNA which contains roughly centrally thebase pairs to be mutated. Both ACA and GCA were used as a complementarycodon for the amino acid cystein to be introduced. These oligonucleotideprimers were produced with a commercially available DNA synthesiser. Nodifference was found in the mutation rate between the two codons forcystein.

In order to produce the proteins according to the invention, thetransformed recombinant micro-organism is cultivated and then theprotein according to the invention is separated, cleaned up andconcentrated from the culture.

The production method for the proteins according to the invention willnow be illustrated by way of example with reference to theabovementioned transfected escherichia coli K13 strain JM105.

The transfected bacteria contain the gene sequence for the alteredinterferon-γ and a trc promotor inducible by IPTG. The bacteria aretaken in cultures and expression of the mutated interferon-γ is inducedby addition of IPTG. The expressed altered interferon-γ is stored by thebacteria cells in so-called "inclusion bodies". In order to clean theexpressed altered interferon-γ, the bacteria cells after successfulexpression are opened and the "inclusion bodies" are freed of solublebacterial proteins by multiple washing. The opening is undertakenpreferably mechanically, for example by ultrasound. The proteinaccording to the invention is brought into solution by a denaturing stepwith guanidine hydrochloride and separated. Then the protein accordingto the invention is renatured by dilution in a phosphate buffer, andfolded into its biologically active form. The interferon-γ, in this waypure to more than 90%, is obtained at a rate of up to 30% of the totalprotein content of the E.coli culture. It can then be concentrated andfurther purified by an affinity chromatography on a column or in abatch-like process and after a further filtration step, e.g. an HPLC-gelfiltration, achieves a purity of more than 95%.

A batch-like process in the sense of this invention, also known as abatch method, is understood to mean the following: the chromatographicmaterial is regularly stirred into a solution of the protein accordingto the invention in such a way that the altered interferon-γ isuniformly distributed and bonds to the chromatographic material and theinterferon-γ protein concentration comes to no more than about 2 mg/mlpacked chromatographic material. The chromatographic material laden withinterferon-γ is washed in the batch with phosphate buffer and thealtered interferon-γ is then eluated in the batch with a saline solutionin the phosphate buffer. In this case for example, SP-sepharose orAffi-gel-blue can be used as chromatographic material.

The figures show:

FIG. 1: the amino acid sequence of the human recombinant humaninterferon-γ (SEQ ID NO: 1);

FIG. 2: the amino acid sequence of the variant of the recombinant humaninterferon-γ according to claim 1 (SEQ ID NO: 2);

FIG. 3: the amino acid sequence of the variants of the recombinant humaninterferon-γ according to claim 2; and

FIG. 4: the base sequence of the oligonucleotides, which were used toproduce the recombinant DNA which codes for the proteins according tothe invention.

Some embodiments of the invention, given by way of example, will beexplained in the following.

EXAMPLE 1

The amino acids Glu8 and Ser70 were exchanged according to the followingmethod and a correspondingly altered interferon-γ was produced.

The interferon-γ gene of the plasmid pKK-233-2/IFN-γ was recloned in thephages M13mp18. For this purpose a plasmid preparation was digested withthe restriction endonucleases EcoRI and HindIII. By means of thisdigestion, the vector was split into two fragments with the lengths 751base pairs and 4305 base pairs. Both fragments were separated from oneanother via a 1% agarose gel, and the corresponding bands, of a size of751 base pairs, were cut out from the gel and isolated. This fragmentcontained, in addition to the complete interferon-γ-gene, also thetrc-promoter and further vector ingredients at the 5' end.

A 150 ml culture of escherichia coli TG1 was infected with the phageM13mp18. After incubation for 5 hours, the cells were centrifuged off,resuspended once and again pelletised. The double-strand circular phageDNA was cleaned up by the Quiagen™ plasmid isolation kit (DiagenCompany) from the cells. The RF-DNA of the phage M13mp18 was likewisedigested with the restriction enzymes EcoRI and HindIII and cleaned viaa 1% agarose gel. Then the isolated EcoRI/HindIII insert, 751 bp long,was cloned from the vector pKK-233-2/IFN-γ in the phages M13mp18.

In order to produce expression vectors for the new variant of theinterferon-γ, mutations of the DNA sequence were carried out at specificpoints. The method is based on hybridisation of an oligonucleotide withthe desired target sequence on single-strand DNA (ssDNA) of the phageM13 to be altered. In order to produce the two spot mutations of Glu8and Ser70 to cystein, the following oligonucleotides were used: for themutation of Glu8 to Cys - TAAGGTTTTC TGCACATTTT ACATATGGG 29(SEQ IDNO:4) - or - TAAGGTTTTC TGCGCATTTT ACATATGGG 29(SEQ ID NO:5) - and formutation of Ser70 to Cys - TGATGGTCTC CACACACTTT TGGATGCTC 29(SEQ IDNO:6) - oder - TGATGGTCTC CACGCACTTT TGGATGCTC 29(SEQ ID NO:7)

The oligonucleotides were produced with the aid of a commerciallyavailable synthesiser. The two codons for cystein are TGT and TGC. Thecorresponding complementary base sequence in the nucleotide strand isGCA or ACA. Both codons were used (emphasised). As a length foroligonucleotide primers, that of 29 base pairs was selected. All thenucleotides had to be phosphorylised at their 5' end withT4-polynucleotidekinase for the mutagenesis.

Independently of the sequence of the oligonucleotides, hybridisation wascarried out at 80° C. in a water bath. After execution of the in vitromutagenesis, the transformation of the double-strand RF form of thephage DNA was carried analogously to a normal plasmid with the CaCl₂method. Cells of E.coli were added to the recombinant phage-RF-DNA, IPTGand X-Gal and plated out. After overnight incubation at 37° C. fourteenphage clones were respectively picked from each mutagenesis reaction andagain incubated together with E.coli for five hours at 37° C.

The sequence monitoring of the mutated cDNA of the bacteriophages, whichis intended to code for the new variant of the recombinant interferon-γ,was effected by didesoxy-sequencing, with the T7-sequencing kit ofPharmacia. Thus the desired mutations were confirmed.

Following the mutagenesis of the phage-DNA, the sequence 751 bp long,which coded for the altered rhu-IFN-γ, was cut out from the phage DNAwith the aid of the same restriction endonucleases, EcoRI and HindIIIand cloned back into the phages pKK233-2. The bacterium escherichia coliK12 strain JM105 was then transfected with this plasmid.

Expression of the altered interferon-γ was then carried out according tothe methods described above. The biological, antiviral activity of thealtered interferon-γ was determined by the reduction in thevirus-induced cytopathic effect which resulted after treatment with thealtered interferon-γ. Human lung fibroblasts A549 were used as a cellstrain, and as a virus the encephalomyocarditis virus.

Analysis of the altered interferon-γ obtained showed that its biologicalactivity was unaltered in comparison to recombinant human interferon-γ.In contrast to recombinant human interferon-γ with a melting temperatureof 52.5° C., the melting temperature of the new variant was considerablyincreased at 68.5° C. With an identical biological activity, thereconsequently resulted for the new variant of interferon-γ a considerablyimproved thermal stability in comparison to unaltered interferon-γ.

EXAMPLE 2

A variant of the recombinant human interferon-γ was produced in whichthe two amino acids Ala18 and His112 were exchanged for cystein.Production of the corresponding DNA, phage DNA, plasmid DNA and thecorresponding expression organisms was carried out as in Example 1. Thefollowing oligonucleotides were used for hybridisation of the ssDNA: forthe mutation of Alal8 to Cys - CATCTGAATG ACCGCAATTA AAATATTTC 29(SEQ IDNO:8) - or - CA TCTGAATG ACCACAATTA AAATATTTC 29(SEQ ID NO:9) - and formutation of His112 to Cys - ACTTGGATGA GTTCGCATAT TGCTTTGCG 29(SEQ IDNO:10) - or - ACTTGGATGA GTTCACATAT TGCTTTGCG 29(SEQ ID NO:11)

In this variant of recombinant human interferon-γ also there was anincreased thermal stability with a melting point of the interferon-γ of78.6° C. with simultaneous indication of antiviral activity of the newvariant.

EXAMPLE 3

A variant of the recombinant human interferon-γ was produced in whichthe two amino acids Lys81 and Leu121 were exchanged for cystein.Production of the corresponding DNA, plasmid-DNA, phage-DNA and of thecorresponding expression micro-organism was effected as described inExample 1. The following oligonucleotides were used to produce thevariant: for the mutation of Lys81 to Cys - TGCTATTG-KA AAAACAGACATTCATGTCT 29(SEQ ID NO:12) - or - TGCTATTGAA AAAGCAGACA TTCATGTCT 29(SEQID NO:13) - and for mutation of Leu121 to Cys - TAGCTGCTGG CGAACATTCAGCCATCACT 29(SEQ ID NO:14) - or - TA GCTGCTGG CGAGCATTCA GCCATCACT29(SEQ ID NO:15) - for the mutation of Gln49 to Cys - AAAAGGAGACA.ATGCAGCTC TGCATTATT 29(SEQ ID NO:16) - or - AAAAGGAGAC AATACAGCTCTGCATTATT 29(SEQ ID NO:17)

This variant of the recombinant human interferon-γ revealed a biologicalantiviral activity similar to that of the unaltered recombinantinterferon-γ with improved thermal stability.

EXAMPLE 4

A variant of the recombinant interferon-γ was produced in which the twoamino acids Gln49 and Leu96 were exchanged for cystein. Production ofthe corresponding DNA, plasmid-DNA, phage-DNA and of the correspondingexpression micro-organism was carried out likewise as described inExample 1. The following oligonucleotides were used to produce thisvariant of interferon-γ: for the mutation of Gln49 to Cys - AAAAGGAGACAATGCAGCTC TGCATTATT 29(SEQ ID NO:16) - or - AAAAGGAGAC AATACAGCTCTGCATTATT 29(SEQ ID NO:17) - and for mutation of Leu96 to Cys -CCGAATAATT AGTGCACTTT TCGAAGTCA 29(SEQ ID NO:18) - or - CCGAATAATTAGTACACTTT TCGAAGTCA 29(SEQ ID NO:19)

For this variant of interferon-γ also the advantages illustrated in theprevious Examples resulted. In this case also this variant ofinterferon-γ with an antiviral activity which corresponds to about 30%of the unaltered recombinant human interferon-γ, is suitable for theapplications according to the invention as a therapeutic agent or as afine chemical such for example as for the stimulation of cell cultures.

EXAMPLE 5

A variant of the altered recombinant human interferon-γ shown in Example1 was produced, in which the amino acids Glu8 and Ser70 were exchangedfor cystein, and in which in addition, in an otherwise known way, theamino acid sequence of the monomeric polypeptide C-terminal wasshortened by 10 amino acids.

With increased thermal stability, this variant of the recombinant humaninterferon-γ reveals an activity which lies 1-4 times higher than theantiviral activity of the unaltered recombinant human interferon-γ. Thusa variant of interferon-γ is presented which, in addition to increasedthermal stability, simultaneously shows increased biological activity.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 19                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 143                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - -  - - <400> SEQUENCE: 1                                                    - - Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Le - #u Lys Lys Tyr Phe        Asn                                                                               1               5 - #                 10 - #                 15             - - Ala Gly His Ser Asp Val Ala Asp Asn Gly Th - #r Leu Phe Leu Gly Ile                   20     - #             25     - #             30                  - - Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Ly - #s Ile Met Gln Ser Gln               35         - #         40         - #         45                      - - Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys As - #n Phe Lys Asp Asp Gln           50             - #     55             - #     60                          - - Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Gl - #u Asp Met Asn Val Lys       65                 - # 70                 - # 75                 - # 80       - - Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp As - #p Phe Glu Lys Leu Thr                       85 - #                 90 - #                 95              - - Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Ar - #g Lys Ala Ile His Glu                  100      - #           105      - #           110                  - - Leu Ile Gln Val Met Ala Glu Leu Ser Pro Al - #a Ala Lys Thr Gly Lys              115          - #       120          - #       125                      - - Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Ar - #g Arg Ala Ser Gln              130              - #   135              - #   140                          - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 144                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Xaa = Glu or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (18)                                                          <223> OTHER INFORMATION: Xaa = Ala or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (49)                                                          <223> OTHER INFORMATION: Xaa = Gln or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (70)                                                          <223> OTHER INFORMATION: Xaa = Ser or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (81)                                                          <223> OTHER INFORMATION: Xaa = Lys or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (96)                                                          <223> OTHER INFORMATION: Xaa = Leu or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (112)                                                         <223> OTHER INFORMATION: Xaa = His or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (121)                                                         <223> OTHER INFORMATION: Xaa = Leu or Cys                                      - - <400> SEQUENCE: 2                                                         - - Met Gln Asp Pro Tyr Val Lys Xaa Ala Glu As - #n Leu Lys Lys Tyr Phe        1               5 - #                 10 - #                 15              - - Asn Xaa Gly His Ser Asp Val Ala Asp Asn Gl - #y Thr Leu Phe Leu Gly                   20     - #             25     - #             30                  - - Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Ar - #g Lys Ile Met Gln Ser               35         - #         40         - #         45                      - - Xaa Ile Val Ser Phe Tyr Phe Lys Leu Phe Ly - #s Asn Phe Lys Asp Asp           50             - #     55             - #     60                          - - Gln Ser Ile Gln Lys Xaa Val Glu Thr Ile Ly - #s Glu Asp Met Asn Val       65                 - # 70                 - # 75                 - # 80       - - Xaa Phe Phe Asn Ser Asn Lys Lys Lys Arg As - #p Asp Phe Glu Lys Xaa                       85 - #                 90 - #                 95              - - Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gl - #n Arg Lys Ala Ile Xaa                  100      - #           105      - #           110                  - - Glu Leu Ile Gln Val Met Ala Glu Xaa Ser Pr - #o Ala Ala Lys Thr Gly              115          - #       120          - #       125                      - - Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gl - #y Arg Arg Ala Ser Gln          130              - #   135              - #   140                          - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 143                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (8)                                                           <223> OTHER INFORMATION: Xaa = Glu or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (18)                                                          <223> OTHER INFORMATION: Xaa = Ala or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (49)                                                          <223> OTHER INFORMATION: Xaa = Gln or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (81)                                                          <223> OTHER INFORMATION: Xaa = Lys or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (96)                                                          <223> OTHER INFORMATION: Xaa = Leu or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (70)                                                          <223> OTHER INFORMATION: Xaa = Ser or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (112)                                                         <223> OTHER INFORMATION: Xaa = His or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (121)                                                         <223> OTHER INFORMATION: Xaa = Leu or Cys                                     <220> FEATURE:                                                                <221> NAME/KEY: SITE                                                          <222> LOCATION: (135)..(143)                                                  <223> OTHER INFORMATION: Some or all of these - #residues may be missing            beginning with the c-terminal end - #of the protein.                     - - <400> SEQUENCE: 3                                                         - - Met Gln Asp Pro Tyr Val Lys Xaa Ala Glu As - #n Leu Lys Lys Tyr Phe        1               5 - #                 10 - #                 15              - - Asn Xaa Gly His Ser Asp Val Ala Asp Asn Gl - #y Thr Leu Phe Leu Gly                   20     - #             25     - #             30                  - - Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Ar - #g Lys Ile Met Gln Ser               35         - #         40         - #         45                      - - Xaa Ile Val Ser Phe Tyr Phe Lys Leu Phe Ly - #s Asn Phe Lys Asp Asp           50             - #     55             - #     60                          - - Gln Ser Ile Gln Lys Xaa Val Glu Thr Ile Ly - #s Glu Asp Met Asn Val       65                 - # 70                 - # 75                 - # 80       - - Xaa Phe Phe Asn Ser Asn Lys Lys Lys Arg As - #p Asp Phe Glu Lys Xaa                       85 - #                 90 - #                 95              - - Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gl - #n Arg Lys Ala Ile Xaa                  100      - #           105      - #           110                  - - Glu Leu Ile Gln Val Met Ala Glu Xaa Ser Pr - #o Ala Ala Lys Thr Gly              115          - #       120          - #       125                      - - Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gl - #y Arg Arg Ala Ser              130              - #   135              - #   140                          - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 4                                                         - - taaggttttc tgcacatttt acatatggg         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 5                                                         - - taaggttttc tgcgcatttt acatatggg         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 6                                                         - - tgatggtctc cacacacttt tggatgctc         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 7                                                         - - tgatggtctc cacgcacttt tggatgctc         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 8                                                         - - catctgaatg accgcaatta aaatatttc         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 9                                                         - - catctgaatg accacaatta aaatatttc         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 10                                                        - - acttggatga gttcgcatat tgctttgcg         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 11                                                        - - acttggatga gttcacatat tgctttgcg         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 12                                                        - - tgctattgaa aaaacagaca ttcatgtct         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 13                                                        - - tgctattgaa aaagcagaca ttcatgtct         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 14                                                        - - tagctgctgg cgaacattca gccatcact         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 15                                                        - - tagctgctgg cgagcattca gccatcact         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 16                                                        - - aaaaggagac aatgcagctc tgcattatt         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 17                                                        - - aaaaggagac aatacagctc tgcattatt         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 18                                                        - - ccgaataatt agtgcacttt tcgaagtca         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 19                                                        - - ccgaataatt agtacacttt tcgaagtca         - #                  - #                29                                                                    __________________________________________________________________________

We claim:
 1. A substituted interferon-gamma (IFN-γ) proteincomprising:(a) a polypeptide comprising the amino acid sequence of SEQID NO: 2 whereinXaa at position 8 is Glu or Cys, Xaa at position 70 isSer or Cys, Xaa at position 18 is Ala or Cys, Xaa at position 112 is Hisor Cys, Xaa at position 81 is Lys or Cys, Xaa at position 121 is Leu orCys, Xaa at position 49 is Gln or Cys, and Xaa at position 96 is Leu orCys,wherein Cys residues are present at both positions of at least onepair selected from the group consisting of positions 8 and 70, 18 and112, 81 and 121, and 49 and 96; (b) a polypeptide which differs from apolypeptide according to (a) by the deletion of 1 to 10 amino acids fromthe C-terminus; or (c) a homodimer of a polypeptide according to (a) or(b).
 2. A DNA molecule comprising a sequence that codes for apolypeptide according to claim
 1. 3. A recombinant vector, comprising aDNA sequence that codes for a polypeptide according to claim
 1. 4. Therecombinant vector of claim 3, wherein the vector is a plasmid.
 5. Therecombinant vector of claim 4, wherein the plasmid is pKK233-2.
 6. Therecombinant vector of claim 3, wherein the vector is a bacteriophage. 7.The recombinant vector of claim 6, wherein the bacteriophage is M13mp18.8. A recombinant micro-organism, transformed with a vector according toone of claims 3 to
 7. 9. The recombinant micro-organism of claim 8,wherein the micro-organism is a bacterium.
 10. The recombinantmicro-organism of claim 9, wherein the bacterium is Escherichia coli K12strain JM105.
 11. A method of producing the recombinant bacteriophage ofclaim 6 or 7, said method comprising the steps of digesting plasmidpKK233-2 containing an IFN-γ-encoding insert with a restrictionendonuclease, inserting the resulting DNA section coding for IFN-γ intothe DNA of bacteriophage M13mp18, and modifying the IFN-γ codingsequence to encode said substituted IFN-γ polypeptide.
 12. The method ofclaim 11, wherein the plasmid is digested with the restrictionendonucleases EcoRl and HindIII.
 13. A method of producing a recombinantbacterium comprising the step of transfecting a bacterial cell with theplasmid of claim 4 or
 5. 14. A method of producing the polypeptide ofclaim 1, said method comprising the steps of cultivating a bacterium,transformed with a vector according to one of claims 3 to 7, breakingopen said transformed bacterium and isolating the polypeptide.
 15. Themethod of claim 14, wherein the bacteria cells are disruptedmechanically.
 16. The method of claim 14 wherein the isolation of thepolypeptide comprises the steps of washing the bacterial cell vesicleswhich contain the polypeptide, solubilizing the polypeptide withguanidinium hydrochloride, diluting the resulting solution, andrenaturing the polypeptide.
 17. The method of claim 16, wherein thepolypeptide is further isolated by column or batch affinitychromatography followed by gel filtration.
 18. The method of claim 17,wherein batch affinity chromatography is conducted by bringing anaffinity matrix into uniform contact with a solution containing thepolypeptide, washing the matrix, and eluting the polypeptide from thematrix in batch format.
 19. The method of claim 18, wherein the affinitychromatography matrix is SP-SEPHAROSE™ or AFFI-GEL BLUE™.
 20. A methodof treating a patient having at least one of a kidney rumor, chronicgranulomatosis and neurodermatitis, said method comprising the step ofadministering to said patient an effective amount of a compositioncomprising a substituted IFN-γ protein according to claim 1.